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    • br Discussion Regulation of redox homeostasis

    2021-11-11


    Discussion Regulation of redox homeostasis is critical in the maintenance of normal cell functions, and both glutathione S-transferase and peroxiredoxin enzymes are important contributors to this process. There are numerous reports of aberrant expression patterns of GSTP linked with cancer and with altered response of tumor cells to a variety of anticancer drugs (reviewed in [21]). The identification of single-nucleotide polymorphisms in GSTP1-1 (Ile/Val105, Ala/Val114) has led to attempts to correlate their incidence with disease susceptibility and drug metabolism. For example, the GSTP1-1B allele was found to associate with bladder and testicular cancer, and decreased GSTP1-1A was linked with prostate cancer, but associations between GSTP1-1 polymorphisms and colorectal or lung cancer have not been found [18], [22]. In a similar vein, increased expression of Prdx6 has been associated with malignancies of various organ sites including breast [23], and Prdx6 supports the growth, invasiveness, and metastasis of breast cancer cells [24]. Moreover, the general peroxidase and PLA2 activities of Prdx6 promote growth and metastases of cancer cells [25], [26]. Although epidemiological studies remain inconsistent, based on the protein interactions that regulate responses to reactive oxygen species, activation of Prdx6 as a peroxidase initially depends on its heterodimerization with GSTP1-1 [2], [3], [6]. S-glutathionylation of target proteins is a function recently ascribed to GSTP1-1 [27] and Prdx6 is one of the enzymes for which S-glutathionylation serves to enhance enzyme activity [8]. The in silico modeling (Fig. 7) and deletion studies suggest that the sites of closest contact between Prdx6 and the GSTP1-1 monomers are on the interface of their interaction at residues 115–124 for GSTP1-1 and 163–169 for Prdx6 [6]. Adding a GSTP1-1 synthetic peptide (aa 113–130) into the mixture of Prdx6 and GSTP1-1 results in a substantial inhibition of heterodimerization of these proteins (∼80%) [6]. Amino Indacaterol Maleate 114 (Ala for GSTP1-1A or GSTP1-1B or Val for GSTP1-1C or GSTP1-1D) is in this site of interaction and could be critical in stabilizing the complex. Another GSTP1-1 synthetic peptide (aa 98–112) also inhibits GSTP1-1 and Prdx6 heterodimerization by∼25% [6]. This site contains amino acid 105 (Ile in GSTP1-1A and GSTP1-1D or Val in GSTP1-1B and GSTP1-1C), which also influences protein binding [6]. Such observations imply that each site of the GSTP1-1 allelic variation (aa 105 and 114) may be involved in its binding to Prdx6. Most probably, the combination of differences in molecular volume (Ala, 69Å3; Val, 120Å3; and Ile, 204Å3) and hydrophobicity in the side chains will influence the affinity of GSTP1-1 for Prdx6. Our present data show that affinities of GSTP1-1A (KD 51nM) or GSTP1-1C (KD 57nM) for Prdx6 are higher than those of GSTP1-1B (KD 101nM) or GSTP1-1D (KD 94nM). The affinity of binding determines proximity between a catalytic Cys47-sulfenate of Prdx6 and activated GSH (thiolate) bound to the GSTP1-1 allelic variant and, consequently, the efficiency with which the sulfenate at Cys47 is S-glutathionylated and reduced (i.e., activated) [6]. Our data show that the peroxidase activities of MCF-7 cell lysates transiently transfected with GSTP1-1A or GSTP1-1C were substantially higher than those of the same cells transfected with either GSTP1-1B or GSTP1-1D. The catalytically inactive Y7F mutant of GSTP1-1A did not support activation of Prdx6 (Table 1) and furthermore peroxidase activity toward phospholipid hydroperoxide was abolished by the addition of specific Prdx6 inhibitor MJ33 [6]. The interface of the Prdx6–GSTP1-1 heterodimer is essentially similar to that of the GSTP1-1 homodimer (Fig. 7). E. coli-expressed and purified GSTP1-1A has better catalytic efficiency and greater affinity for CDNB (Km=0.33±0.07mM) than GSTP1-1B (Km=1.15±0.07mM) [28]. Because the active unit of GSTP1-1 is a homodimer [6], this result may be explained by differences in interactions between monomeric subunits, similar to those for the Prdx6–GSTP1-1 heterodimer shown here. As a further corollary, in earlier studies the bulkiness of aa 105 (Ile/Ala, Ile/Val, Ile, and Ile/Trp) was shown to correlate negatively with GSTP1-1 kcat/Km values for anti-diol epoxides [29].